PROJECT SUMMARY/ABSTRACT This is a competing renewal application studying the biological basis of clinical phenotype and outcome of biliary atresia, the most common cause of neonatal cholestasis. The disease results from a fibro-inflammatory obstruction of extrahepatic bile ducts and present in early infancy. Despite nearly uniform progression to end- stage cirrhosis, the variable response to surgical/medical treatment and rate of progression of disease suggest the existence of unrecognized biological processes that are driving different phenotypes or stages of disease. In the previous tenure of the award, we found evidence of increased signaling via IL-8, TNF, and components of the inflammasome in pathogenesis of bile duct injury, and the simultaneous activation of molecular circuits dependent on IL-33 to induce tissue repair. We also identified a key role for MMP-7 in bile duct epithelial injury and as a highly sensitive and specific biomarker for biliary atresia. In preparation for this application, we applied computer modeling and high analytics to mine the hepatic transcriptome and found a 14-gene signature that predicts 2-year survival with the native liver and identifies glutathione metabolism as a new therapeutic target to suppress fibrosis. Using serum proteomics, we also uncovered serum proteins that segregate with children with advanced fibrosis as determined by portal hypertension. These data form the foundation for the new studies proposed in three inter-related aims: 1) To discover molecular determinants of outcome and pathogenesis of biliary atresia, 2) To identify biomarkers of portal hypertension during progression of liver disease, and 3) To define pathogenic mechanisms of tissue injury in biliary atresia. Experiments for Aim 1 will use RNAseq data from a large cohort to mine gene groups and molecular pathways that predict clinical outcome, followed by complementary studies in mouse models of biliary atresia and neonatal fibrosis in pre-clinical trials to suppress fibrosis by targeting metabolic circuits in the liver. Experiments for Aim 2 will use data from serum proteomics to investigate how SEMA6B, sFRP3, COMMD7, VCAM1, and BMX perform as biomarkers of portal hypertension individually or in combination. And experiments in Aim 3 will derive biliary organoids from the liver of subjects with biliary atresia and test hypothesis related to defects of cell maturation and to how the activation of fibrogenesis in cholangiocytes is an important mechanisms of bile duct injury. By applying highly complementary approaches to study tissues from adequately sized cohorts that have been phenotyped prospectively, our experiments will provide insight into new biomarkers of disease, their role in pathogenesis, and how new clinical trials can be personalized based on biological end-point.